Many flaviviruses, such as Dengue virus, Japanese encephalitis virus, yellow fever virus, and West Nile virus, are arthropod-borne human pathogens. These viruses are of global concern for major epidemics that can cause millions of infecfions and many deaths each year. Currently, there is no commercially available vaccine, and treatment relies on supportive therapy. The flavivirus mature particles are small enveloped viruses with a diameter of about 50 nm. The viral pari;icles consist of an electron-dense nucleocapsid core of about 30 nm surrounded by a host-derived lipid bilayer and 180 copies of hwo glycoproteins (envelope protein E and membrane protein M) forming an icosahedral shell. The flaviviruses undergo dramafic conformational changes during the maturafion from immature to mature particles, and from the pre-fusion niature particle state to the post-fusion state during infecfion and membrane fusion with host cell. Currently, there are only atomic resolution structures ofthe soluble fragment of E, prM protein and capsid protein. The structure ofthe enfire virus particles is based on cryo-electron microscopy (cryo-EM) 3-D reconstrucfions that are limited to low-to-intermediate resolufions (30-9.5 A). In collaborafion with other participants of this PPG applicafion, we plan to apply and develop the single particle cryo-EM image processing and 3-D reconstrucfion techniques to solve the flavivirus mature and immature particles to near-atomic resolutions (4-5 A) and to construct backbone models ofthe entire E and M proteins in the icosahedral virus environment. We also plan to determine the first 3-D structure of a fusion intermediate of flavivirus to about 10 A resolufion, thus filling the remaining gap of structural knowledge of the major steps in the flavivirus assembly, maturafion, and infection processes. With these studies, we aim to understand the structural basis of flavivirus assembly and infection and hopefully help the development of effective vaccines and treatments of the flavivirus infecfions. ' RELEVANCE (See instructions): Flaviviruses infect millions of people each year. Detailed structural informafion will be gathered for the Flaviviruses at their major steps of viral particle assembly, maturafion and infecfion processes. The new knowledge will enhance our understanding ofthe underiying mechanism of Flavivirus life cycle and contribute to the design of effecfive intervenfion strategies. The cryo-EM method developed here can also be used to study many other human viruses, such as herpes virus, norovirus, hepatifis C virus, etc.